1. Field of the Invention
This invention relates generally to gender determination of bird eggs. More specifically, this invention relates to egg gender determination by analysis of sex-specific compounds in allantoic fluid of the egg by ion mobility spectrometry (IMS).
2. Related Art
A common method for sexing birds is the Japanese method of vent sexing which involves manual procedures and requires trained professionals with highly specialized skills. Another common method involves feather sexing of chicks which carry the a slow feathering gene. However, birds which carry this slow feathering gene also exhibit slower growth and reduced egg production. There are other methods, such as illumination of the egg to determine eye color at day 10, polymerase chain reaction (PCR), immunoassay, and DNA based techniques. All of these techniques have problems with speed, cost effectiveness, or negative production effects.
For example, U.S. Pat. No. 5,377,618 (Crews et al.) and U.S. Pat. No. 5,648,468 (Spaulding) disclose methods for urging the sex of avian embryos. Crew et al. uses an injection of estradiol-17 beta for female development and dihydrotestosterone for male development. Spaulding uses isolated sex-specific proteins to create antibodies for immunoassays and for injection back into the hen to create sex selection pressure.
Also, U.S. Pat. No. 5,508,165 (Halverson et al. ""165), U.S. Pat. No. 5,679,514 (Baker), and U.S. Pat. No. 5,707,809 (Halverson et al. ""809) disclose methods for determining the sex of avian embryo by DNA techniques.
Ion mobility spectrometry (IMS) offers a technique that can provide reliable results quickly, uses current genetic strains, and is less traumatic to the birds. IMS also may be integrated into current hatchery process equipment (i.e. vaccination equipment) as an automated technique that is labor-saving and cost effective. With the invented technique based on IMS, the sex of resulting birds can be determined prior to hatching from the egg, allowing the poultry industry to realize significant cost and labor savings while processing tens of millions of eggs per day.
According to the invention, during the incubation process, the outer hard shells of eggs are drilled and samples of allantoic fluid are removed. This process does not intrude into the embryo or amniotic fluid. The allantoic fluids are directly introduced into an ion mobility spectrometer (IMS) for analysis, for example, of estradiol or other sex-specific compound. The resulting spectra contain the relevant marker peaks with unique mobilities that are sex-specific. In the negative mode, peaks with reduced mobilities around 2.0 were found in eggs with female embryos and peaks with reduced mobilities from 1.5 to 1.25 were found in eggs with male embryos. In the positive mode, there were peaks at reduced mobilities of about 1.6 found in eggs with male embryos and peaks with reduced mobilities of about 1.4 found in eggs with female embryos. There were also sex-specific peaks at lower intensities at other reduced mobilities. Based on these data, sex determination may be made based on the presence or absence of one or several of these peaks or combinations of these peaks. As may be seen, sex determination may also be accomplished in either the positive or negative mode of IMS.
Allantoic fluids were analyzed directly by direct injection of microliter quantities into the IMS source region. The small quantities needed for analysis translate well into an online process which may be integrated with existing inoculation equipment. Sample preparation in this preferred method is minimal to non-existent, thus enhancing sample throughput. The egg fluid may be able to be analyzed quickly and without sample preparation by direct injection of the fluid into the IMS source region.